Familial Advanced Sleep Phase Syndrome (FASPS) is the only known Mendelian phenotype of the human circadian system. We've identified and characterized the clinical phenotype and identified five genes that, when mutated, cause FASPS. Two of these, casein kinase I? & ? (CKI?/?), are recognized to harbor mutations that segregate with FASPS in families and lead to decreased activity in vitro. A mutation in a third gene, period 2 affects a CKI?/? phosphorylation site. Work in a number of laboratories has characterized some substrates of these kinases, but a comprehensive and unbiased method for identifying substrates has been impossible given the large number of kinases and phosphatases present in any cell or organism. We will employ an innovative chemical genetic approach to specifically label substrates of these enzymes by engineering mutations into the ATP binding pocket. Reciprocal chemical modifications of ATP are engineered to synthesize ATP analogs that can only be accommodated by the mutated (analog-sensitive) kinases. This will provide a more complete compendium of substrates for CKI?/? and will allow assessment of the redundant and unique functions of each enzyme. This approach will also be applied in identifying multiple phosphorylation sites on known substrates by these kinases. In vitro biochemical assays can be performed to monitor specific effects of the FASPS mutations on each of these substrates. Next, transgenic mice will be generated to carry a BAC with each gene harboring the analog-sensitive mutations. These will be crossed onto null backgrounds and will represent mice with near normal kinase activity since the analog-sensitive kinases still accept, and transfer phosphate groups from ATP. Mice carrying analog sensitive mutations for both CKI? and CKI? will be generated. We can then rapidly and reversibly inactivate these kinases through use of chemical inhibitors that bind specifically in the analog-sensitive ATP binding site. These mice will be studied at different developmental time points to monitor the phenotype when one or both kinases are inactivated. In particular, we will focus on the circadian system but are also interested in whether the lethality that is seen in the CKI? knock out mice is the result of its effects on development or of its activity throughout the life of the mouse. This work will result in identification of many CKI?/? substrates and molecular dissection of the role of these kinases in human circadian rhythmicity. Identification of substrates and dissection of particular pathways in phenotypes such as circadian rhythmicity will have profound implications for therapeutics of circadian phenotypes and understanding of physiological mechanisms. [unreadable] PUBLIC HEALTH RELEVANCE: CKI? and CKI? are important kinases for many essential biological functions. This proposal outlines a plan to elucidate the normal role of CKI? and CKI? through identification of their substrates and studies aimed at understanding substrates that are important for the functional consequences of CKI?/? in circadian rhythm. We will also examine phenotypes resulting from reversibly inactivating these kinases in vivo. [unreadable] [unreadable] [unreadable]